Kinetics of activation of a PKC-regulated epithelial calcium channel

Abstract

The kinetics of calcium entry through regulated calcium channels in cultured renal proximal tubule cells was studied with Fura-2 fluorescence ratio imaging in single cells. The calcium entry was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol-12-myristat-13-acetate (PMA), similar to that observed for activation by osmo-mechanical stress. OAG (2.5μM) or PMA (0.5μM) activated calcium entry is characterized by a significant latency between agonist application and the response, whereas the effect of osmo-mechanical stress was immediate. This pre-response latency was 260 ± 70s with OAG stimulation and 79.2 ± 17.3s with PMA stimulation. Once a cell responds, the intracellular calcium level reaches a peak value within seconds. The cell response to agonist is independent of the response of neighboring cells. The response kinetics resembles those of the calcium sparks in excitable cells, except the response is much slower. In all cases, the response appears to be an all-or-none event, that is characteristics of an elementary binary switch. It is suggested that the binary response and the lack of coordinated response of calcium entry in single cells results from limited availability of the calcium channels and/or PKC that activates the channel. The experimental data could be fit to a single binary response mathematical model assuming each response reflected an elementary event of a single channel opening or a co-ordinated opening of a cluster of several channels.